Pseudorabies (Aujeszky's disease) is a highly infectious disease of domestic animals, including swine, cattle, sheep, dogs, cats, mink and rats, caused by pseudorabies virus (PRV), a member of the herpes virus family (Roizman, et al., Intervirology 16:201(1981)). Swine are the primary host for this virus and piglets under five weeks of age are most susceptible. Adult pigs often become latently infected following exposure to PRV only to have the virus reactivated later in time.
The disease is characterized by severe respiratory illness, abortions, reduced litter size, decreased growth rate and an often fatal encephalitis (Gustafson, in Diseases of Swine, Dunn and Ledman, Eds., Iowa State Press, 1975). Current control measures include vaccination with either inactivated or attenuated PRV or test and removal procedures (See Gustafson, supra, (1975).
Modified live virus (MLV) and inactivated whole virus vaccines have been used extensively as a source to induce immunity against many diseases. Modified live virus stocks are generally produced by multiple passages in permissive or semi-permissive cell lines. One common feature of high passage virus stocks is that they are usually less virulent or attenuated.
PRV vaccines have been produced by a variety of methods. However, the MLV forms of vaccination result in the virus being maintained in the environment. Thus, complete eradication of the virus is impossible. An alternative vaccination approach would be the use of an inactivated recombinant vaccine expressing selected immunogenic PRV glycoproteins. The development of such a vaccine requires a knowledge of the organization of the PRV genome and the glycoproteins it encodes.
The PRV genome consists of a double-stranded DNA molecule with a molecular weight of approximately 90 kilo daltons (Kd) (Rubenstein and Kaplan, Virology 66:385(1975)). The genome is separated by inverted repeat sequences into a unique short (U.sub.S) and a unique long (U.sub.L) region (Stevely, J. Virol. 22:232(1977); Ben-Porat, et al., Virology 95:285(1979)). Viral glycoproteins have been shown to be encoded by genes located in both the U.sub.S and U.sub.L regions of the genome. It has been found that in at least two strains of attenuated PRV (Norden and Bartha strains; Norden Laboratories, Lincoln, Nebr.), the attenuation appears to be directly correlated with a genomic deletion. (Petrovskis, et al., J. Virol. 60:1166(1986)). The deletion is usually about 2-4 thousand base pairs in length and is located in the U.sub.S region of the genome. At least one protein, gI, is encoded by this deleted region.
Hampl, et al., J. Virol. 52:583(1984) have described five glycoproteins that are incorporated into the envelope of PRV. These include gI (130 Kd), gII a,b,c (125 Kd, 74 Kd, 58 Kd), gIII (98 Kd), gIV (98 Kd), and gV (62 Kd). Another viral encoded glycoprotein referred to as gX (98 Kd) has been shown to accumulate in the medium of infected cells (Rea, et al., J. Virol. 54:21(1985)). Two other viral glycoproteins have been identified: gp50 (Wathen and Wathen, J. Virol. 51:57(1984) and gp63 (Petrovskis, et al., J. Virol. 59:216(1986)). The map locations of five of the glycoproteins have been reported. The gII complex and gIII have been shown to map in the U.sub.L region between map positions 0.105-0.130 and near map position 0.40, respectively (Mettenleiter, et al., Virology 152:66(1986); Robbins, et al., J. Virol. 58:339(1986)). gX, gp50, gI, and gp63 have been mapped in the U.sub.S region of the genome. (Rea, et al., supra, (1985); Wathen and Wathen, supra, (1984); Mettenleiter, et al., J. Virol. 53:52(1985); Petrovskis, et al., supra, (1986)). (See FIG. 1.)
In the Norden and Bartha strains, flanking the gI deletion on the upstream side, i.e., the start of the gene, are the two glycoproteins gp50 and gp63. On the downstream side, i.e., the end of the gene, is the region of DNA involved with replication. Sera from animals exposed to natural infections contain antibody to gp50, gp63 and gI. Animals vaccinated with modified live virus do not contain antibody to gI.
Several monoclonal antibodies have been produced that neutralize PRV in vitro. Hampl, et al., supra, (1984) reported that monoclonal antibodies directed against gIII effectively neutralize the virus. Monoclonal antibodies specific for gI and gII also have virus neutralizing activity (J. Rziha, personal communication; Mettenleiter, et al., supra, (1986). Wathen and Wathen, supra, (1984), reported that monoclonal antibodies raised against gp50 have virus neutralizing activity. Additionally, it has been shown that mice passively immunized with monoclonal antibodies directed against gp50 are protected following challenge with PRV.
Various attempts have been made to develop a useful vaccine for immunization of animals against PRV infection. Use of the PRV glycoprotein, gp50, as a potential subunit vaccine candidate has been reported by Marchioli, et al., J. Virol. 61:3977(1987). Petrokovis, et al., Patent Cooperation Treaty (PCT) Application WO 87/02058 reports the production of subunit vaccines for PRV using one of gI, gp50 and gp63 polypeptides. Production of an attenuated PRV vaccine, comprising DNA from PRV is reported by Shih, et al., PCT Application WO 87/01287. An attenuated PRV vaccine is produced using a sequence of PRV essential for replication of the attenuated virus from which a portion of the repeat sequence has been deleted.
Modification of the live PRV by use of a temperature-sensitive PRV strain has also been reported, U.S. Pat. No. 4,514,497. The production of PRV glycoprotein genes in procaryotic and eucaryotic expression systems and their use as immunogens is described by Robbins, et al., European Patent Application 0 162 738.
Recently, numerous reports have demonstrated the utility of using vaccinia virus recombinants expressing foreign viral genes as vaccines. Successful expression and immunization have been reported with infectious vaccinia virus recombinants including those containing the genes for hepatitis B surface antigen (Smith, et al., Nature 302:490(1983); Paoletti, et al., Proc. Natl. Acad. Sci. USA 81:193(1984), influenza virus hemagglutinin (Panicali, et al., Proc. Natl. Acad. Sci. USA 80:5364(1983); herpes simplex glycoprotein D (Paolettl, et al., supra, (1984); Cremer, et al., Science 228:737(1985); rabies glycoprotein G (Wiktor, et al., Proc. Natl. Acad. Sci. USA 81:7194(1984)), vesicular stomatitis glycoprotein G (Mackett, et al., Science 227:433(1985), and human respiratory syncytial virus G glycoprotein (Ball, et al., Proc. Natl. Acad. Sci, USA 83:246(1986); Elango, et al., Proc. Natl. Acad. Sci. USA 83:1906(1986)).